The NF-kappaB/Rel family of transcription factors play an important role in inflammatory responses. In nonstimulated cells, the heterodimeric NF-kappaB complex is located in the cytoplasm, where it is associated with IkappaBalpha, an inhibitory molecule. In response to stimulation by a number of agents, including the pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1, IkappaBalpha undergoes phosphorylation and subsequent proteolytic degradation, allowing the p50/p65 NF-kappaB heterodimer to migrate to the nucleus and transactivate various inducible target genes. A protein kinase complex, termed IkappaB kinase (IKK), plays a pivotal role in the phosphorylation IkappaBalpha in response to cytokine stimulation. Protein prenylation may participate in NF-kappaB activation, although that role in not fully understood. Isoprenylation inhibitors alter proper membrane localization and biological activity of various signaling intermediates, including members of the Ras family of GTP-binding proteins. In this study, we examined the effect of manumycin A, a potent and selective farnesyltransferase inhibitor, on the activation of NF-kappaB through signaling mechanisms that control the IKK pathway. Treatment of the cells with 10 uM manumycin A inhibited NF-kappaB transactivation and nuclear translocation of p65/RelA in response to TNF. Manumycin also blocked TNF-induced IKK-dependent phosphorylation of IkappaB alpha. Using solid-phase kinase assays, it was determined that activation of the multisubunit IKK complex and an upstream kinase, NF-kappaB inducing kinase (NIK), by TNF was rapidly attenuated after cell treatment with manumycin A. Unexpectedly, the inhibition of endogenous farnesyltransferase activity was associated with an increase in the phosphatidylinositol (PI) 3-kinase/Akt cascade and p38 MAP kinase activity but not JNK/SAPK. However, pharmacological inhibition of PI 3-kinase and p38 MAP kinase with wortmannin/LY294002 and SB202190, respectively, did not prevent the inactivation of TNF signaling elicited by manumycin A both in the human hepatoblastoma (HepG2) and Chinese hamster ovary cells. Subsequently, it was shown that manumycin A blocked the phosphorylation and proteolytic degradation of IkappaBalpha upon cell stimulation with a second cytokine, interleukin-1beta. On the basis of these data we propose the participation of short-lived farnesylated proteins in cytokine-mediated activation of the IKK pathway leading to NF-kappaB dependent transcriptional activity. Whether manumycin interferes with the recruitment and/or formation of signaling competent multiprotein complex at the cell membrane is not known. As proinflammatory cytokines play an important role in the pathogenesis of insulin resistance, our findings suggest that inhibition of cytokine signaling at a converging step upstream of IKK may represent a potential target for new strategies to improve insulin-resistant state.